The matrix level of pyrophosphate (PPi) in mitochondria isolated
from etiolated pea (Pisum sativum L. cv. Alaska) stems
was evaluated, on the basis of an enzymatic assay, to be approx.
0.2 mM. Pyrophosphate could enter from the cytoplasm
to the mitochondria via adenine nucleotide translocase
(ANT), because F– and Ca21 (two penetrating PPiase inhibitors)
and atractylate (ANT inhibitor) inhibited PPiase activity
in isolated mitochondria supplied with PPi. This result
was also confirmed by measuring oxygen consumption and
membrane potential (DY) in succinate-energized mitochondria.
In a medium free of phosphate (Pi), the addition of PPi
before the substrate rendered possible an ADP-stimulated
oxygen consumption that was inhibited by F– or Ca21. In a similar experiment, ADP induced the dissipation of DY when
it was added after the succinate-generated DY had reached a
steady state and, again, F– inhibited this dissipation. These
results imply that PPi enters the mitochondria where it is
hydrolyzed to 2 Pi which become available for the H1-ATPase
(EC 3.6.1.34). In addition, PPi may be synthesized by the
H1-PPiase (EC 3.6.1.1), acting as a synthase. This evidence
arises from the observation that Pi stimulated an oxygen consumption
(respiratory control ratio of 1.7) that was inhibited
by F– or Ca21. The physiological role of the mitochondrial
H1-PPiase is discussed in the light of the consideration that
this enzyme can catalyse a readily reversible reaction.
